An understanding of the human polyomavirus, JC virus, is of great importance due to its active infection of immunocompromised persons. Active infection results in progressive multifocal leukoencephalopathy (PML). PML causes the destruction of myelin resulting in dementia, coma, and death. The intracellular pathway of JC virus has not been determined, providing a barrier to the treatment of active infection. The goal of this investigation is to determine how JC and other polyomaviruses are transported in cells. This includes determining the vesicles used for transport and the motor proteins that drive the motion. I propose that the transport of specific populations of vesicles is independent of the cargo contained. This hypothesis will be tested in two steps: 1. The human, simian, and murine polyomaviruses will be imaged during intracellular transport to determine which vesicles and motor proteins are used. Two- and three-color fluorescence imaging will be used to determine which vesicles the virus uses for transport. Characterization of the motor proteins involved in transport will be based on the individual steps of the virus on the cytoskeleton. These experiments will be the first to make use of a recently developed technique, Fluorescence Imaging with One Nanometer Accuracy (FIONA), to probe viral transport on the level of individual motor protein steps. 2. The experimental techniques developed will then be extended to non-viral cargo to test the general relationship between transport dynamics and vesicle population. To this end the polyomaviruses will serve as probes of the three major endocytic pathways. Non-viral cargo will be loaded into specific vesicles and imaged during intracellular transport. The dynamics observed will be compared to those of the polyomaviruses. Overall, this research is expected to contribute significantly to our understanding of three interrelated fields; viral infection, imaging technology, and intracellular transport. [unreadable] [unreadable] [unreadable]